Clinical features of the patient
A 64-year-old woman with kidney stones and hydronephrosis was admitted to Nanjing Drum Tower Hospital on April 22, 2018. After percutaneous nephrolithotomy on April 24, the patient had high fever with a body temperature of 40.7°C and signs of septic shock. Her white blood cell count was 15.8 × 109/L; neutrophil percentage, 97.1%; C-reactive protein level, 65.9 mg/L; and procalcitonin, 4.94 ng/mL.
Bacterial culture and isolation
A urine sample and two sets of blood samples were cultured for bacterial identification. Gram-negative bacilli were identified on microscopic examination after 18-h culture of the blood samples in aerobic and anaerobic bottles. The samples were also cultured on a sheep blood agar plate (BAP) at 35°C in a 5% CO2 atmosphere overnight, and a slow-growing, needle-tip-like small colony was identified on the BAP. We named the three strains of Gram-negative colonies isolated from blood culture as EC1, EC2, and EC3. EC1 and the needle-tip-like EC2 were isolated from the same aerobic culture bottle, while EC3 was isolated from the anaerobic culture bottle. The urine sample was cultured on sheep BAP at 35°C in a 5% CO2 atmosphere. After 24 h of incubation, >10,0000 CFU/ml of Gram- negative colonies were detected on the BAP. The strains identified in the blood and urine samples were isolated for further characterization.
Treatment and outcome
The patient was transferred to the ICU for replenishment of blood volume by treatment with a vascular active medicine, and also received oxygen inhalation treatment. For anti-infection therapy, the patient was treated with imipenem (500 mg, Q8H) for one week. Based on elevated blood creatinine level (136 mmol/L) and oliguria, acute renal injury was diagnosed and the patient received renal replacement therapy. The patient recovered well and was discharged on May 9. The study protocol was approved by the Ethics Committee of Nanjing Drum Tower Hospital.
Colony morphology analysis and Gram staining
The morphological characteristics of strains EC1, EC2, and EC3 were compared in three different culture conditions: aerobic, 5% CO2, and anaerobic. We also observed the morphological characteristics of the strains at three time points: after 24, 48, and 72 h of culture. The EC1, EC2, and EC3 bacterial cells were also analyzed by Gram staining. E. coli ATCC 25922 was used as the control strain.
Identification of strains
The three strains, EC1, EC2, and EC3, were identified using the VITEK 2 system (bioMérieux, France) and the API 32E system (bioMérieux, France) according to the manufacturer’s instructions. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (micro Typer MS) was used to confirm the identification. The colonies isolated from urine were identified by the VITEK 2 system. The biochemical characteristics of the strains were examined with the oxidation fermentation experiment and oxidase test.
Antimicrobial susceptibility testing
Antimicrobial susceptibility testing was performed using the VITEK 2 system with the GN13 card and the disk diffusion method (Oxoid Lid, UK). For the disk diffusion method, a 0.5 McFarland suspension of each of the three strains was prepared and spread on Mueller Hinton (MH) agar and cultured in a 5% CO2 environment for 16–18 h with the following antimicrobials: ampicillin, ampicillin-sulbactam, amikacin, ceftazidime, cefotaxime, cefuroxime, cefoxitin, imipenem, piperacillin-tazobactam, cefepime, levofloxacin, and sulfamethoxazole-trimethoprim. The minimum inhibitory concentration (MIC) and disk diffusion method results were determined based on the criteria for testing Enterobacteraccording to CLSI M100 (Clinical and Laboratory Standards Institute 2018).
Auxotrophy testing
Auxotrophy tests for thymidine, hemin, thiamine, and menadione were performed as described previously (Wellinghausen N et al. 2009). The final concentration of the four supplements was 10 µg/ml in MH agar. EC1, EC2, EC3, and ATCC25922 were inoculated on the MH agar and incubated at 35°C in 5% CO2 for 48 h.
Preparation of genomic DNA and WGS analysis
Genomic DNA of the three strains was extracted from a culture grown overnight in Luria-Bertanimedium at 35°C under agitation (220 rpm) using the QIAamp DNA minikit (Qiagen, Hilden, Germany). Library preparations were constructed following the manufacturer’s protocol (Illumina TruSeq DNA Nano Library Prep Kit) and sequenced using the IlluminaHiseq PE150 platform to generate 450-bp paired-end reads.Trimmed sequencing reads were subjected to de novo assemblywith SPAdes pipeline version 3.9.0 (Bankevich A et al. 2012) under the default settings. Multi-locus sequence typing (MLST) was performed according to a previously published method (Larsen MV et al. 2012). WGS was performed to identify genes that contained previously reported causative mutations for the E. coli SCV phenotypes and the nucleotide similarity was analyzed by CLC Genomics Workbench 21.0.3. The genome sequences of EC1, EC2, and EC3 have been deposited in GenBank under accession numbers QVHR00000000, QVHQ00000000, and QVHS00000000, respectively.
PCR analysis
Extracted DNA was amplified by PCR under standard conditions as described previously (Sahuquillo-Arce JM et al. 2017) with the following primers for amplification of the 510-bp fragment of the can gene (which is not present in capnophilic strains): canF (5′-TTGGTGGCGGTAACATCCAG-3′) and canR (5′-GAAACTGGCACAAGCGC AAA-3′). The following PCR protocol was used: 5 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 55°C, and 1 min at 72°C; and a final extension of 7 min at 72°C. The amplification products were separated by electrophoresis on an agarose gel with 2% ethidium bromide staining, and the size of the amplicons was determined based on their homology with a molecular weight marker (PCR marker; Promega Corporation, Madison, WI, USA) DL2000.